Efficient production of γ-aminobutyric acid using engineered Escherichia coli whole-cell catalyst.

γ-Aminobutyric acid (GABA) has been widely used in the food, feed, pharmaceutical, and chemical industry fields. Previously, we developed a whole-cell catalyst capable of converting L-glutamate (L-Glu) into GABA by overexpressing the glutamate decarboxylase gene (gadz11) from Bacillus sp. Z11 in Escherichia coli BL21(DE3). However, to enhance cell permeability, a freeze-thaw treatment is required, and to enhance GADZ11 activity, pyridoxal 5'-phosphate (PLP) must be added to the reaction system. The aim of this study is to provide a more efficient approach for GABA production by engineering the recombinant E. coli above. First, the inducible expression conditions of the gadz11 in E. coli were optimized to 37 °C for 6 h. Next, an ideal engineered strain was produced via increasing cell permeability by overexpressing sulA and eliminating PLP dependence by constructing a self-sufficient system. Furthermore, an efficient whole-cell biocatalytic process was optimized. The optimal substrate concentration, cell density, and reaction temperature were 1.0 mol/L (the molecular ratio of L-Glu to L-monosodium glutamate (L-MSG) was 4:1), 15 and 37 °C, respectively. Finally, a whole-cell bioconversion procedure was performed in a 3-L bioreactor under optimal conditions. The strain could be reused for at least two cycles with GABA yield, productivity and conversion ratio of 206.2 g/L, 117.8 g/L/h and 100.0%, respectively. This is currently the highest GABA productivity from a mixture of L-Glu and L-MSG reported without the addition of cofactors or additional treatment of cells. This work demonstrates that the novel engineered E. coli strain has the potential for application in large-scale industrial GABA production.

SARS-CoV-2 spike-specific TFH cells exhibit unique responses in infected and vaccinated individuals.

Long-term humoral immunity to SARS-CoV-2 is essential for preventing reinfection. The production of neutralizing antibody (nAb) and B cell differentiation are tightly regulated by T follicular help (TFH) cells. However, the longevity and functional role of TFH cell subsets in COVID-19 convalescents and vaccine recipients remain poorly defined. Here, we show that SARS-CoV-2 infection and inactivated vaccine elicited both spike-specific CXCR3+ TFH cell and CXCR3- TFH cell responses, which showed distinct response patterns. Spike-specific CXCR3+ TFH cells exhibit a dominant and more durable response than CXCR3- TFH cells that positively correlated with antibody responses. A third booster dose preferentially expands the spike-specific CXCR3+ TFH cell subset induced by two doses of inactivated vaccine, contributing to antibody maturation and potency. Functionally, spike-specific CXCR3+ TFH cells have a greater ability to induce spike-specific antibody secreting cells (ASCs) differentiation compared to spike-specific CXCR3- TFH cells. In conclusion, the persistent and functional role of spike-specific CXCR3+ TFH cells following SARS-CoV-2 infection and vaccination may play an important role in antibody maintenance and recall response, thereby conferring long-term protection. The findings from this study will inform the development of SARS-CoV-2 vaccines aiming to induce long-term protective immune memory.

Co-Fe3C pair sites catalyst with heterometallic dual active sites for efficient oxygen reduction reaction.

Newly emerging metal-based pair sites catalysts show great potential because they can provide more metal active centers with synergistic effect for green catalysis, compared with single site catalysts. However, both the synthesis and catalytic mechanisms of the pair sites catalyst with new structural features need to be developed vigorously to promote the desired chemical reactions, especially carbon-based metal catalysts for green energy storage and conversion devices. Herein, we constructed highly active Co-Fe3C pair sites on N-doped graphite catalyst (CNCo-Fe3C) by a two-step strategy, which have electron interactions of heterometallic atoms and can play better synergistic effect. X-ray absorption spectra and density functional theory (DFT) calculation further identify the presence of heterometallic active sites in the pair sites catalyst, resulting in electron redistribution and positive d-band center due to the electron interactions. The more positive d-band center model predicts the optimization of the adsorption energy of oxygen-containing intermediates, and reduces the energy barrier of the determining step. This further results in superior oxygen reduction reaction (ORR) performance with a half-wave potential of 0.90 V versus reversible hydrogen electrode (vs.RHE) and superior long-term stability for about 20 h with only 2.3 % decrease at 0.75 V vs.RHE in 0.1 M KOH solution. Additionally, it also shows significant peak power density of 124 mW cm-2 and prominent cycling stability performance exceeding 400 h at 5 mA cm-2 in the Zn-air battery (ZAB) test, which is higher than that of Pt/C catalyst. This work provides a new idea for the regulation of intrinsic activity of non-noble metal ORR catalysts through the synergistic effect of the pair sites.

Production of N-acetylglucosamine from carbon dioxide by engineering Cupriavidus necator H16.

The conversion of CO2 into valuable bioactive substances using synthetic biological techniques is a potential approach for mitigating the greenhouse effect. Here, the engineering of C. necator H16 to produce N-acetylglucosamine (GlcNAc) from CO2 is reported. First, GlcNAc importation and intracellular metabolic pathways were disrupted by the deletion of nagF, nagE, nagC, nagA and nagB genes. Second, the GlcNAc-6-phosphate N-acetyltransferase gene (gna1) was screened. A GlcNAc-producing strain was constructed by overexpressing a mutant gna1 from Caenorhabditis elegans. A further increase in GlcNAc production was achieved by disrupting poly(3-hydroxybutyrate) biosynthesis and the Entner-Doudoroff pathways. The maximum GlcNAc titers were 199.9 and 566.3 mg/L for fructose and glycerol, respectively. Finally, the best strain achieved a GlcNAc titer of 75.3 mg/L in autotrophic fermentation. This study demonstrated a conversion of CO2 to GlcNAc, thereby providing a feasible approach for the biosynthesis of various bioactive chemicals from CO2 under normal conditions..

Surface Modification of Hollow Structure TiO2 Nanospheres for Enhanced Photocatalytic Hydrogen Evolution.

Engineering the surface structure of semiconductor is one of the most promising strategies for improving the separation and transfer efficiency of charge, which is a key issue in photocatalysis. Here, we designed and fabricated the C decorated hollow TiO2 photocatalysts (C-TiO2), in which 3-aminophenol-formaldehyde resin (APF) spheres were used as template and carbon precursor. It was determined that the C content can be easily controlled by calcinating the APF spheres with different time. Moreover, the synergetic effort between the optimal C content and the formed Ti-O-C bonds in C-TiO2 were determined to increase the light absorption and greatly promote the separation and transfer of charge in the photocatalytic reaction, which is verified from UV-vis, PL, photocurrent, and EIS characterizations. Remarkably, the activity of the C-TiO2 is 5.5-fold higher than that of TiO2 in H2 evolution. A feasible strategy for rational design and construction of surface-engineered hollow photocatalysts to improve the photocatalytic performance was provided in this study.

Platinum-Cobalt Nanowires for Efficient Alcohol Oxidation Electrocatalysis.

The compositions and surface facets of platinum (Pt)-based electrocatalysts are of great significance for the development of direct alcohol fuel cells (DAFCs). We reported an approach for preparing ultrathin PtnCo100-n nanowire (NW) catalysts with high activity. The PtnCo100-n NW alloy catalysts synthesized by single-phase surfactant-free synthesis have adjustable compositions and (111) plane and strain lattices. X-ray diffraction (XRD) results indicate that the alloy composition can adjust the lattice shrinkage or expansion of PtnCo100-n NWs. X-ray photoelectron spectroscopy (XPS) results show that the electron structure of Pt is changed by the alloying effect caused by electron modulation in the d band, and the chemical adsorption strength of Pt is decreased, thus the catalytic activity of Pt is increased. The experimental results show that the activity of PtnCo100-n for the oxidation of methanol and ethanol is related to the exposed crystal surface, strain lattice and composition of catalysts. The PtnCo100-n NWs exhibit stronger electrocatalytic performance for both methanol oxidation reaction (MOR) and ethanol oxidation reaction (EOR). The dominant (111) plane Pt53Co47 exhibits the highest electrocatalytic activity in MOR, which is supported by the results of XPS. This discovery provides a new pathway to design high activity, stability nanocatalysts to enhance direct alcohol fuel cells.

Strained Lattice Gold-Copper Alloy Nanoparticles for Efficient Carbon Dioxide Electroreduction.

Electrocatalytic conversion of carbon dioxide (CO2) into specific renewable fuels is an attractive way to mitigate the greenhouse effect and solve the energy crisis. AunCu100-n/C alloy nanoparticles (AunCu100-n/C NPs) with tunable compositions, a highly active crystal plane and a strained lattice were synthesized by the thermal solvent co-reduction method. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) results show that AunCu100-n/C catalysts display a subtle lattice strain and dominant (111) crystal plane, which can be adjusted by the alloy composition. Electrochemical results show that AunCu100-n/C alloy catalysts for CO2 reduction display high catalytic activity; in particular, the Faradaic efficiency of Au75Cu25/C is up to 92.6% for CO at -0.7 V (vs. the reversible hydrogen electrode), which is related to lattice shrinkage and the active facet. This research provides a new strategy with which to design strong and active nanoalloy catalysts with lattice mismatch and main active surfaces for CO2 reduction reaction.

Monoclonal antibodies constructed from COVID-19 convalescent memory B cells exhibit potent binding activity to MERS-CoV spike S2 subunit and other human coronaviruses.

Introduction: The Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are two highly contagious coronaviruses causing MERS and COVID-19, respectively, without an effective antiviral drug and a long-lasting vaccine. Approaches for diagnosis, therapeutics, prevention, etc., particularly for SARS-CoV-2 that is continually spreading and evolving, are urgently needed. Our previous study discovered that >60% of sera from convalescent COVID-19 individuals, but <8% from general population, showed binding activity against the MERS-CoV spike protein, indicating that SARS-CoV-2 infection boosted antibodies cross-reactive with MERS-CoV. Methods: To generate antibodies specific to both SARS-CoV-2 and MERS-CoV, here we screened 60 COVID-19 convalescent sera against MERS-CoV spike extracellular domain and S1 and S2 subunits. We constructed and characterized monoclonal antibodies (mAbs) from COVID-19 convalescent memory B cells and examined their binding and neutralizing activities against human coronaviruses. Results and Discussion: Of 60 convalescent serum samples, 34 showed binding activity against MERS-CoV S2, with endpoint titers positively correlated with the titers to SARS-CoV-2 S2. By sorting single memory B cells from COVID-19 convalescents, we constructed 38 mAbs and found that 11 mAbs showed binding activity with MERS-CoV S2, of which 9 mAbs showed potent cross-reactivity with all or a proportion of spike proteins of alphacoronaviruses (229E and NL63) and betacoronaviruses (SARS-CoV-1, SARS-CoV-2, OC43, and HKU1). Moreover, 5 mAbs also showed weak neutralization efficiency against MERS-CoV spike pseudovirus. Epitope analysis revealed that 3 and 8 mAbs bound to linear and conformational epitopes in MERS-CoV S2, respectively. In summary, we have constructed a panel of antibodies with broad-spectrum reactivity against all seven human coronaviruses, thus facilitating the development of diagnosis methods and vaccine design for multiple coronaviruses.

Molecular characteristic and pathogenicity analysis of a novel multiple recombinant ALV-K strain.

Avian leukosis virus (ALV) can induce various tumors and cause serious production problems. ALVs isolated from chickens were divided into six subgroups (A-J). In 2012, a strain of a putative novel subgroup of ALVs was isolated from Chinese native chickens in Jiangsu Province and named as ALV-K. In this study, three ALV-K strains (JS14LH01, JS13LH14, and JS15SG01) were isolated from chickens with suspected ALV infection in Jiangsu Province. Their complete genomes were amplified, sequenced, and analyzed systematically. The results showed that JS14LH01 and JS13LH14 were ALV-K and ALV-E recombinant strains. Whereas JS15SG01 is an ALV-K, ALV-E, and ALV-J multiple recombinant strain containing the U3 region of ALV-J. The pathogenicity test of JS15SG01 revealed that, compared with previous ALV-K strains, the viremia and viral shedding level of JS15SG01-infected chickens were significantly increased, reaching 100 % and 59 %, respectively. More important, JS15SG01 induced significant proliferation of gliocytes in the cerebral cortex of infected chickens, accompanied by the neurotropic phenomenon. This is the first report about a multiple recombinant ALV-K strain that could invade and injure the brain tissue of chickens in China. Our findings enriched the epidemiologic data of ALV and helped to reveal the evolution of ALV strains prevalent in chicken fields.

Molecular characterization of avian leukosis virus subgroup J in Chinese local chickens between 2013 and 2018.

Avian leukosis virus subgroup J (ALV-J) was first isolated from broiler chickens in China in 1999; subsequently, it was rapidly introduced into layer chickens and Chinese local chickens. Recently, the incidence of ALV-J in broiler and layer chickens has significantly decreased. However, it has caused substantial damage to Chinese local chickens, resulting in immense challenges to their production performance and breeding safety. To systematically analyze the molecular characteristics and the epidemic trend of ALV-J in Chinese local chickens, 260 clinical samples were collected for the period of 2013-2018; 18 ALV-J local chicken isolates were identified by antigen-capture enzyme-linked immunosorbent assay and subgroup A-, B-, and J-specific multiplex PCR. The whole genomic sequences of 18 isolates were amplified with PCR and submitted to GenBank. Approximately, 55.5% (10/18) of the 18 isolates demonstrated a relatively high homology (92.3-95.4%) with 20 ALV-J early-isolated local strains (genome sequences obtained from GenBank) in gp85 genes clustering in a separated branch. The 3' untranslated region (3' UTR) of the 18 isolates showed a 195-210 and 16-28 base pair deletion in the redundant transmembrane region and in direct repeat 1, respectively; 55.5% (10/18) of the 18 isolates retained the 147 residue E element. The U3 gene of 61.1% (11/18) of the 18 isolates shared high identity (94.6-97.3%) with ALV-J early-isolated local strains. These results implied that the gp85 and U3 of ALV-J local chicken isolates have rapidly evolved and formed a unique local chicken branch. In addition, it was determined that the gene deletion in the 3'UTR region currently serves as a unique molecular characteristic of ALV-J in China. Hence, the obtained results built on the existing ALV-J molecular epidemiological data and further elucidated the genetic evolution trend of ALV-J in Chinese local chickens.

The Bipartite Sequence Motif in the N and C Termini of gp85 of Subgroup J Avian Leukosis Virus Plays a Crucial Role in Receptor Binding and Viral Entry.

Subgroup J avian leukemia virus (ALV-J), belonging to the genus Alpharetrovirus, enters cells through its envelope surface unit (gp85) via specifically recognizing the cellular receptor chicken Na+/H+ exchanger type I (chNHE1), the 28 to 39 N-terminal residues of which were characterized as the minimal receptor functional domain in our previous studies. In this study, to further clarify the precise organization and properties of the interaction between ALV-J gp85 and chNHE1, we identified the chNHE1-binding domain of ALV-J gp85 using a series of gp85 mutants with segment substitutions and evaluating their effects on chNHE1 binding in protein-cell binding assays. Our results showed that hemagglutinin (HA) substitutions of amino acids (aa) 38 to 131 (N terminus of gp85) and aa 159 to 283 (C terminus of gp85) significantly inhibited the interaction between gp85 and chNHE1/chNHE1 loop 1. In addition, these HA-substituted chimeric gp85 proteins could not effectively block the entry of ALV-J into chNHE1-expressing cells. Furthermore, analysis of various N-linked glycosylation sites and cysteine mutants in gp85 revealed that glycosylation sites (N6 and N11) and cysteines (C3 and C9) were directly involved in receptor-gp85 binding and important for the entry of ALV-J into cells. Taken together, our findings indicated that the bipartite sequence motif, spanning aa 38 to 131 and aa 159 to 283, of ALV-J gp85 was essential for binding to chNHE1, with its two N-linked glycosylation sites and two cysteines being important for its receptor-binding function and subsequent viral infection steps.IMPORTANCE Infection of a cell by retroviruses requires the attachment and fusion of the host and viral membranes. The specific adsorption of envelope (Env) surface proteins to cell receptors is a key step in triggering infections and has been the target of antiviral drug screening. ALV-J is an economically important avian pathogen that belongs to the genus Alpharetrovirus and has a wider host range than other ALV subgroups. Our results showed that the amino acids 38 to 131 of the N terminus and 159 to 283 of the C terminus of ALV-J gp85 controlled the efficiency of gp85 binding to chNHE1 and were critical for viral infection. In addition, the glycosylation sites (N6 and N11) and cysteines (C3 and C9) of gp85 played a crucial role in the receptor binding and viral entry. These findings might help elucidate the mechanism of the entry of ALV-J into host cells and provide antiviral targets for the control of ALV-J.

Isolation and molecular characterization of the first subgroup J avian leukosis virus from chicken in Pakistan.

Since subgroup J avian leukosis virus (ALV-J) was first isolated in the United Kingdom in 1988, it has seriously hindered the development of the poultry industry worldwide. Although cases of ALV-J infection have been reported as early as 2001 in Pakistan, there was no further research on the isolation and molecular characteristics of ALVs. In the present study, we first isolated two ALVs from suspicious clinical samples that were collected from a desi chicken farm in Pakistan. The results of multiplex PCR and indirect immunofluorescent antibody assays confirmed that the two isolates (PK19FA01 and PK19SA01) belonged to ALV-J. The complete genomes of the two isolates were amplified, sequenced, and systematically analyzed. We found that gp85 of PK19FA01 was more similar to that of the prototype strain HPRS103, whereas gp85 of PK19SA01 was more similar to that of American strains. The two isolates contained an intact E element of 147 residues and had a unique 135 bp deletion in the redundant transmembrane of the 3' untranslated region. The U3 region of the two isolates was highly homologous to that of American ALV-J strains. To our knowledge, this is the first report of the isolation, complete genome sequencing, and systematic molecular epidemiological investigation of ALV-J in Pakistan. Our findings could enrich epidemiological data and might contributed to more effective measures to prevent and control avian leukosis in Pakistan.

[MiR-124-3p Enhances the Sansitivity of Chronic Myelogenous Leukemia Cell K562-R to Imatinib by Targeting ABCA2].

OBJECTIVE: To investigate the effect and mechanism of miR-124-3p-targeing regulating ABCA2 on chronic myelogenous leukemia cell K562-R. METHODS: CML cells with miR-124-3p-overexpression and ABCA2-over-expression as well as subcutaneoustrans planted tumor nude mice were used as study objects. And the CML cells were divided into four groups: K562-R blank control, miR-124-3p mimic control, ABCA2-overexpression and mimic+PC ABCA2. The effects of miR-124-3p and ABCA2 on CML cells were analyzed. The levels of proliferation-, apoptosis- and autophagy- related protein were determined by Western blot. qRT-PCR was employed to detect the levels of miR-124-3p and ABCA2 in K562-R cells. The relationship between miR-124-3p and ABCA2 was validated by luciferase reporter system assays and bioinformatics. Hoechst/immunohistochemical staining and CCK-8 assay were performed to investigate the function involved. RESULTS: miR-124-3p highly expressed in K562-S cells and lowly expressed in K562-R cells, however, ABCA2 lowly expressed in K562-S cells and highly expressed in K562-R cells. Over-expression of miR-124-3p significantly decreased ABCA2 level and cell growth, but increased autophagy and apoptosis in K562-R cells (P＜0.01). When ABCA2 was over-expressed, the K562-R cell growth was promoted and autophagy and apoptosis were inhibited (P＜0.01). The miR-124-3p promoted cell autophagy and apoptosis but inhibited cell growth in nude mice transplant tumor model (P＜0.01). CONCLUSION: miR-124-3p can target ABCA2 to inhibit the growth of CML cells and promote the cell autophagy and apoptosis of CML cells.

Strain-Modulated Platinum-Palladium Nanowires for Oxygen Reduction Reaction.

Electrocatalytic activity of alloy nanocatalytsts can be manipulated effectively by tuning their physical properties (ensemble, geometric, and ligand effects) to afford optimal surface structure and compositions for proton exchange membrane fuel cell (PEMFC) application. Herein, highly catalytic platinum-palladium nanowires (PtnPd100-n NWs) with a subtle lattice strain and Boerdijk-Coxeter helix type morphology are synthesized through a surfactant-free, thermal single phase solvent method. X-ray diffraction results show that PtnPd100-n NWs are exposed through the (111) facets and their shrinking or expanding lattice parameters can be modulated by the alloy compositions. Electrochemical results reveal that their high catalytic activity correlates with the lattice shrinking, facets, and bimetallic compositions, showing higher activity when the ratio of Pt and Pd is ∼78:22, which is further supported by DFT results. Compared to the nanoparticle type platinum-palladium alloyed catalysts with similar metal compositions (PtnPd100-n NPs), the PtnPd100-n NWs exhibit significantly improved electrocatalytic activity and stability for the oxygen reduction reaction. These findings open new strategies to design the highly active and stable alloy nanocatalysts with controllable compositions.

Development and evaluation of a gp85 protein-based subgroup-specific indirect enzyme-linked immunosorbent assay for the detection of anti-subgroup J avian leukosis virus antibodies.

Avian leukosis virus subgroup J (ALV-J) is an important pathogen for various neoplasms and causes significant economic losses in the poultry industry. Serological detection of specific antibodies against ALV-J infection is important for successful clinical diagnosis. Here, a 293F stable cell line was established to stably express gp85 protein. In this cell line, gp85 protein was expressed at approximately 30 mg/L. A subgroup-specific indirect enzyme-linked immunosorbent assay (iELISA) was developed using ALV-J gp85 protein as coated antigen to detect antibodies against ALV-J. The sensitivity of the iELISA (1:51200 diluted in serum) was 16 times more than that of indirect immunofluorescence assay (IFA; 1:3200 diluted in serum). Moreover, there was no crossreactivity with antibodies against other common avian viruses and other avian leukosis virus subgroups, such as subgroups A and B. The practicality of the iELISA was further evaluated by experimental infection and clinical samples. The results from experimental infection indicated that anti-ALV-J antibodies were readily detected by iELISA as early as 4 weeks after ALV-J infection, and positive antibodies were detected until 20 weeks, with an antibody-positive rate of 11.1% to 33.3%. Moreover, analysis of clinical samples showed that 9.49% of samples were positive for anti-ALV-J antibodies, and the concordance rate of iELISA and IFA was 99.24%. Overall, these results suggested that the subgroup-specific iELISA developed in this study had good sensitivity, specificity, and feasibility. This iELISA will be very useful for epidemiological surveillance, diagnosis, and eradication of ALV-J in poultry farms.

Origin of High Activity and Durability of Twisty Nanowire Alloy Catalysts under Oxygen Reduction and Fuel Cell Operating Conditions.

The ability to control the surface composition and morphology of alloy catalysts is critical for achieving high activity and durability of catalysts for oxygen reduction reaction (ORR) and fuel cells. This report describes an efficient surfactant-free synthesis route for producing a twisty nanowire (TNW) shaped platinum-iron (PtFe) alloy catalyst (denoted as PtFe TNWs) with controllable bimetallic compositions. PtFe TNWs with an optimal initial composition of ∼24% Pt are shown to exhibit the highest mass activity (3.4 A/mgPt, ∼20 times higher than that of commercial Pt catalyst) and the highest durability (<2% loss of activity after 40 000 cycles and <30% loss after 120 000 cycles) among all PtFe-based nanocatalysts under ORR or fuel cell operating conditions reported so far. Using ex situ and in situ synchrotron X-ray diffraction coupled with atomic pair distribution function (PDF) analysis and 3D modeling, the PtFe TNWs are shown to exhibit mixed face-centered cubic (fcc)-body-centered cubic (bcc) alloy structure and a significant lattice strain. A striking finding is that the activity strongly depends on the composition of the as-synthesized catalysts and this dependence remains unchanged despite the evolution of the composition of the different catalysts and their lattice constants under ORR or fuel cell operating conditions. Notably, dealloying under fuel cell operating condition starts at phase-segregated domain sites leading to a final fcc alloy structure with subtle differences in surface morphology. Due to a subsequent realloying and the morphology of TNWs, the surface lattice strain observed with the as-synthesized catalysts is largely preserved. This strain and the particular facets exhibited by the TNWs are believed to be responsible for the observed activity and durability enhancements. These findings provide new insights into the correlation between the structure, activity, and durability of nanoalloy catalysts and are expected to energize the ongoing effort to develop highly active and durable low-Pt-content nanowire catalysts by controlling their alloy structure and morphology.

MiR-125b Suppression Inhibits Apoptosis and Negatively Regulates Sema4D in Avian Leukosis Virus-Transformed Cells.

Subgroup J avian leukosis virus (ALV-J), an oncogenic retrovirus, causes hemangiomas and myeloid tumors in chickens. We previously showed that miR-125b is down-regulated in ALV-J-induced tumors. This study aimed to investigate the possible role of miR-125b in ALV-J-mediated infection and tumorigenesis. Knockdown of miR-125b expression in HP45 cells reduced, whereas over-expression induced late-stage apoptosis. Bioinformatics analysis and luciferase activity assays indicate that miR-125b targets Semaphorin 4D/CD100 (Sema4D) by binding the 3'-untranslated region of messenger RNA (mRNA). Up-regulation of miR-125b in the DF1 cell line suppressed Sema4D expression, whereas miR-125 down-regulation increased Sema4D expression levels. To uncover the function of Sema4D during ALV-J infection, animal infection experiments and in vitro assays were performed and show that Sema4D mRNA levels were up-regulated in ALV-J-infected tissues and cells. Finally, functional experiments show that miR-125 down-regulation and Sema4D over-expression inhibited apoptosis in HP45 cells. These results suggest that miR-125b and its target Sema4D might play an important role in the aggressive growth of HP45 cells induced by avian leukosis viruses (ALVs). These findings improve our understanding of the underlying mechanism of ALV-J infection and tumorigenesis.

Development and application of a colloidal gold test strip for detection of avian leukosis virus.

Avian leukosis virus (ALV) is an avian oncogenic retrovirus that induces leukemia-like proliferative diseases in chickens. ALV infection can result in the development of immunological tolerance and persistent viremia. Since effective vaccines against ALV are not yet available, its current prevention primarily depends on detection and eradication to establish exogenous ALV-free poultry flocks. In this study, a rapid and simple colloidal gold test strip method, specific for the group-specific antigen, p27 protein, was developed and systematically evaluated for the detection of ALV from different samples. The detection limit of this assay was as low as 6.25 ng/ml for p27 protein and 80 TCID50/ml for different subgroups of ALV. Besides, the test strip showed high specificity in the detection of different subgroups of ALV, including ALV-A, ALV-B, ALV-J, and ALV-K, with no cross-reaction with other avian pathogens. Furthermore, we artificially infected specific pathogen-free (SPF) chickens with ALV-J, collected cloacal swabs, and examined viral shedding using both test strips and ELISA. Results from the test strip were highly consistent with that from ELISA. In addition, 1104 virus isolates from anti-coagulant blood samples, 645 albumen samples, and 4312 meconium samples were tested, and the test strip results agreed with those of ELISA kit up to 97.1%. All the results indicated that the colloidal gold test strip could serve as a simple, rapid, sensitive, and specific diagnostic method for eradication of ALV in poultry farms.

Genomic sequence and pathogenicity of the first avian metapneumovirus subtype B isolated from chicken in China.

Avian metapneumovirus (aMPV), which has been reported in many countries, causes an acute upper respiratory tract disease in chickens and turkeys. Although aMPV was first detected in China in 1999, there has been no further effort to isolate and characterize the aMPV subtype B (aMPV/B) from field outbreaks. In the present study, we used Vero cells to culture a viral strain, LN16, isolated from chickens with swollen head syndrome. The results of RT-PCR, indirect immunofluorescent antibody, and G gene sequence analyses confirmed that strain LN16 corresponds to aMPV/B. We amplified and sequenced the complete genome of strain LN16 and found it to be 13,513 nucleotides in length. Nine viral protein genes of the strain were between 93.2% and 98.4% identical to those of the pathogenic field isolate VCO3/60616. However, insertions and deletions were detected in the intergenic regions. Animal experiments showed that 72.7% of chickens infected with strain LN16 had excess mucus, nasal discharge, and inflammation in the lungs and turbinate. In addition, 27.2% of chickens infected with LN16 shed progeny virions. Viral tissue distribution analysis showed that aMPV could be detected in the turbinate and occasionally in immune organs. This is the first report of the isolation of aMPV/B in China and the first complete genome sequence of aMPV/B from chicken. These findings enrich the epidemiological data on aMPV and may contribute to the development of effective measures to prevent its further spread in China.